Date Thesis Awarded
5-2024
Access Type
Honors Thesis -- Access Restricted On-Campus Only
Degree Name
Bachelors of Science (BS)
Department
Biology
Advisor
Lizabeth Allison
Committee Members
Lawrence Evans
Beverly Sher
Mark Forsyth
Abstract
Thyroid hormone is an important component of the endocrine system that regulates growth and metabolism. The hormone circulates in the bloodstream and is transported into target cells, where it then enters the nucleus and binds to tissue-specific thyroid hormone receptors (TRs) to facilitate transcription of thyroid hormone-responsive genes. However, TR is known to rapidly shuttle between the nucleus and cytoplasm. Prior research in our lab has demonstrated more cytoplasmic localization of an acetylation mimic of TR compared to a non-acetylation mimic and wild-type TR. This implies that post-translational modification of TRs via lysine acetylation may be an additional mechanism of regulating thyroid hormone signaling. To investigate whether lysine deacetylase HDAC2 might play a role in TR deacetylation and localization, we transfected HeLa cells with expression plasmids for GFP-tagged TR subtypes, TRα1, β1, and β2. Cells were incubated with either Santacruzamate A—an HDAC2 inhibitor—or a DMSO control post-transfection. After fluorescence imaging and nucleocytoplasmic scoring, cellular localization of TR, measured by N/C ratio, was only significantly different for inhibitor-treated TRβ1 relative to the control. However, the observed shift was towards the nucleus, suggesting an indirect effect due to increased histone acetylation, which led us to conclude that HDAC2 is likely not the primary regulator of TR deacetylation. Resistance to Thyroid Hormone Syndrome (RTH) is a known genetic dysfunction of the thyroid hormone signaling pathway characterized by tissue-specific hypothyroidism. Patients with severe RTH, characterized by shortened stature, mental defects, and bradycardia, have mutations in TRα1, which is specific to skeletal, heart, and brain tissues. Two identified mutations, C392X and E403X, result in premature truncation of TRα1's ligand-binding domain. We hypothesized that these mutations might increase the affinity of mutant TRα1 for nuclear corepressor NCoR1 and enhance nuclear retention. Using cotransfection of expression plasmids for mCherry-tagged TRα1 variants with GFP-NCoR1 or an NCoR1 variant that does not bind TR as a control, we found that the nucleocytoplasmic localization of the mutants was not significantly different in the presence of overexpressed NCoR1 relative to the control, likely because both mutants already show a primarily nuclear localization and enhanced nuclear retention may not be measurable. Thus, we expressed the mutant receptors in HeLa cell lines with NCoR1 knocked out. C392X, but not E403X or wild-type TRα1, demonstrated a significant cytoplasmic shift in knockout cells compared to controls with normal expression of NCoR1, validating our model of impaired NCoR1 release in that mutant. Broadly, these results contribute to general understanding of TR's binding partners within the nucleus and highlight the need for further characterization of the structure and binding dynamics of the E403X mutant and more broadly of protein-protein interactions within endocrine pathways.
Recommended Citation
Danielski, Caroline, "Investigating Protein Interactions Modulating Thyroid Hormone Receptor Localization" (2024). Undergraduate Honors Theses. William & Mary. Paper 2117.
https://scholarworks.wm.edu/honorstheses/2117
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
